Antibodies to the PcrV antigen of Pseudomonas aeruginosa

ABSTRACT

The current invention provides high-affinity antibodies to the  Pseudomonas aeruginosa  PcrV protein that have reduced immunogenicity when administered to treat  Pseudomonas aeruginosa  infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/325,806, filed Dec. 1, 2008, U.S. Pat. No. 8,044,181 issued Oct. 25,2011, which claims benefit of U.S. provisional application No.60/991,679, filed Nov. 30, 2007, each of which applications is hereinincorporated by reference.

BACKGROUND OF THE INVENTION

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen thatrarely causes disease in healthy people, but is a significant problemfor critically ill or immunocompromised individuals. Infection is amajor problem in individuals who have cystic fibrosis (CF), where P.aeorginosa is a causative agent in the progressive loss of lung functionresulting from recurrent and chronic respiratory tract infections withthe bacterium. Others at risk from Pseudomonas aeruginosa infectioninclude patients on mechanical ventilators, neutropenic cancer patients,and burn patients. P. aeruginosa is often resistant to most antibioticsand new treatment approaches are greatly needed.

The type III secretion system is an important virulence factordeterminant in that it inhibits host defense system. Upon activation,the type III secretion apparatus translocates toxins into the cytoplasmof the host cell, resulting in cell rounding, lifting, and cell death bynecrosis or apoptosis. PcrV is an essential component of the type IIIsecretion apparatus. Blocking of PcrV can inhibit toxin secretionthrough the Type III secretion system, thereby allowing naturalclearance mechanisms to eliminate the bacteria. PcrV is conserved in allP. aeruginosa strains tested thus far.

Antibodies to the PcrV antigen of Pseudomonas are known in the art. Thecurrent invention provides improved PcrV antibodies, e.g., for thetreatment of Pseudomonas aeruginosa infections.

BRIEF SUMMARY OF THE INVENTION

The current invention relates to engineered antibodies that bind withhigh affinity, e.g., at about 50 nM, or about 10 nM or less, to the PcrVantigen from Pseudomonas aeruginosa. Such antibodies are oftenfunctional antagonists of the Type III secretion, i.e., the antibodybinds to PcrV and inhibits the Type III secretion system. In someembodiments, an antibody of the invention may bind to PcrV and recruitmultiple cell types of the immune system to stimulate phagocytosis bymacrophages, antibody directed cellular cytotoxicity (ADCC) bymacrophages or NK cells, activation of the complement cascade, and/orgeneration of the oxidative burst by neutrophils.

The antibodies of the invention have variable regions with a high degreeof identity to human germ-line V_(H) and V_(L) sequences and are lessimmunogenic in humans. The CDR3 sequences of the heavy and light chainsof the present invention comprise a pair of binding specificitydeterminants (BSD) from the monoclonal anti-PcrV antibody Mab166 (Franket al., J. Infectious Dis. 186: 64-73, 2002). The antibodies of theinvention typically are capable of competing with Mab166 for binding toa neutralizing epitope on the PcrV protein.

The BSD sequence in CDRH3 has the amino acid sequence NRGDIYYDFTY (SEQID NO:38). The BSD in CDRL3 is FWXTP (where X may be either S or G; SEQID NO:39). Complete V-regions are generated in which the BSD forms partof the CDR3 and additional sequences are used to complete the CDR3 andadd a FR4 sequence. Typically, the portion of the CDR3 excluding the BSDand the complete FR4 are comprised of human germ-line sequences.Preferably, the CDR3-FR4 sequence excluding the BSD differs from humangerm-line sequences by not more than two amino acids on each chain. TheCDR3-FR4 is joined to a V-segment that has a high degree identity, e.g.,at least 80%, or at least 90%, or more to a human germline V segment.

In some aspects, the invention provides an anti-PcrV antibody thatexhibits high affinity binding, e.g., 10 nM or better, to PcrV and is anantagonist of the Type III secretion system.

In many embodiments, an anti-PcrV antibody of the invention thatselectively binds to PcrV comprises: a V_(L) region that comprises aCDR3 comprising FWGTP. In typical embodiments, such an antibody has aV_(L) region V-segment has at least 80% identity to a human germlineV-segment. The FR4 region typically has at least 90% identity to the FR4region of a human germline J segment.

In some embodiments, an anti-PcrV antibody of the invention comprises aCDR3 comprising FWGTP (SEQ ID NO:40), a FR4 and a V-segment, wherein theFR4 comprises at least 90% identity to the FR4 region of the human JK2germline gene segment or at least 90% identity to the JL2 germlinesequence; and the V-segment comprises at least 80% identity to a humangermline Vkappa I or Vkappa III sequence, or at least 80% identity to ahuman germline Vlambda sequence. In some embodiments the V_(L) regionCDR3 has the sequence Q(Q/H)FWGTPYT (SEQ ID NO:41). In some embodiments,the antibody further comprises a V_(H) region that comprises a CDR3having a sequence NRGDIYYDFTY (SEQ ID NO:38), a FR4 and a V-segment,wherein the FR4 comprises at least 90% identity to the FR4 region of thehuman JH3 or human JH6 segment and the V-segment comprises at least 80%identity to the human VH1-18 subclass V-segment or to the human VH3-30.3V segment. In some embodiments, the V_(H) region comprises a CDR3 havinga sequence NRGDIYYDFTYA(M/F)DX₁ (SEQ ID NO:42), wherein X₁ is I, Q, Y,or S.

In further embodiments, the invention provides an anti-PcrV antibodythat binds to PcrV, comprising: a V_(H) region that comprises a CDR3having a sequence NRGDIYYDFTYAMDX₁ (SEQ ID NO:43), wherein X₁ is I, Q,Y, or S; a FR4 and a V-segment, wherein the FR4 comprises at least 90%identity to the FR4 region of the human germline JH3 segment or the FR4region of the human germine JH6 segment, and the V-segment comprises atleast 80% identity to the human germline VH1-18 subclass V-segment or tothe human germline VH3-30.3 subclass V segment, with the proviso thatwhen X₁ is Y, the FR4 region is not WGQGTSVTVSS (SEQ ID NO:44).

In some embodiments, the invention provides an anti-PcrV antibody thatbinds to PcrV, comprising: a V_(H) region that comprises a CDR3 having asequence NRGDIYYDFTYAMDX₁ (SEQ ID NO:43), wherein X₁ is I, Q, Y, or S; aFR4 and a V-segment, wherein the FR4 comprises at least 90% identity tothe FR4 region of the human germline JH3 segment or the FR4 region ofthe human germline JH6 segment, and the V-segment comprises at least 80%identity to the human germline VH1-18 subclass V-segment or to the humangermline VH3-30.3 subclass V segment, with the proviso that when X₁ isY, the FR4 region is not WGQGTSVTVSS (SEQ ID NO:44); and a V_(L) regionthat comprises a CDR3 comprising FW(S/G)TP (SEQ ID NO:39), a FR4 and aV-segment, wherein the FR4 comprises at least 90% identity to the FR4region of the human germline JK2 gene segment or to the FR4 region ofthe human germline JL2 segment; and the V-segment comprises at least 80%identity to the human germline VKI L12 sequence, or at least 80%identity to a Vkappa III sequence, or at least 80% identity to a humangermline Vlambda2 2c or Vlambda3 31 segment.

In some embodiment, the FR4 of the V_(H) region of an antibody of theinvention has the sequence WGQGTX₂VTVSS (SEQ ID NO:45), wherein X₂ is Tor M.

In some embodiments, an antibody of the invention has a light chain CDR3that has the sequence Q(H/Q)FW(G/S)TPYT (SEQ ID NO:46).

In some embodiments, the FR4 of the V_(L) region has the sequenceFGQGTKLEIK (SEQ ID NO:47) or FGGGTKLTVL (SEQ ID NO:48).

The invention also provides an anti-PcrV antibody where the V_(H) regionV-segment has at least 80% identity to the human germline VH3-30.3segment and the heavy chain region CDR1 comprises the sequenceX₃X₄X₅X₆H, wherein X₃ is S, T, or N; X₄ is Y or A; X₅ is A, G, or P; andX₆ is M, I, or L; and the heavy chain region CDR2 comprises the sequenceX₇IX₈YX₉GX₁₀X₁₁X₁₂X₁₃Y(A/I)X₁₄SVKG (SEQ ID NO:49), wherein X₇ is V, F,or N; X₈ is 5 or W; X₉ is D or N; X₁₀ is 5, K, R or Y; X₁₁ is N, 5, D orE; X₁₂ is K, I, or E; X₁₃ is Y, 5, D or W; and X₁₄ is D or S. In someembodiments, the antibody has at least 90% identity to a VH3-30.3 Vsegment. In some embodiments, the CDR1 is TAGMH (SEQ ID NO:50), SYGIH(SEQ ID NO:51), SYGMH (SEQ ID NO:52), SYPLH (SEQ ID NO:53), or NYPMH(SEQ ID NO:54). In some embodiments, the CDR2 is VIWYNGKEISYADSVKG (SEQID NO:55), FISYDGSEKYYASSVKG (SEQ ID NO:56), VISYDGSEKWYADSVKG (SEQ IDNO:57), VIWYDGRNKYYADSVKG (SEQ ID NO:58), VIWYDGYNKDYADSVKG (SEQ IDNO:59), or NIWYDGSSESYIDSVKG (SEQ ID NO:60). In some embodiments, theCDR1 is TAGMH (SEQ ID NO:50), SYGIH (SEQ ID NO:51), SYGMH (SEQ IDNO:52), SYPLH (SEQ ID NO:53), or NYPMH (SEQ ID NO:54); and the CDR2 isVIWYNGKEISYADSVKG (SEQ ID NO:55), FISYDGSEKYYASSVKG (SEQ ID NO:56),VISYDGSEKWYADSVKG (SEQ ID NO:57), VIWYDGRNKYYADSVKG (SEQ ID O:58),VIWYDGYNKDYADSVKG (SEQ ID NO:59), or NIWYDGSSESYIDSVKG (SEQ ID NO:60).

The invention also provides an anti-PcrV antibody where the V_(H) regionV-segment has at least 80% identity, or at least 90% identity, to thehuman germline VH1-18 sub-class V-segment and the CDR1 has the sequenceDHAIS (SEQ ID NO:61) and the CDR2 has the sequence WISPYSGNPNYAQSLQG(SEQ ID NO:62).

The invention also provides an anti-PcrV antibody that binds to PcrV,comprising: a VH region that has a CDR3 sequence NRGDIYYDFTYAFDI (SEQ IDNO:63), a CDR1 sequence DHAIS (SEQ ID NO:61) and a CDR2 sequenceWISPYSGNPNYAQSLQG (SEQ ID NO:62).

In some embodiments the V_(H) region comprises the V-segment region ofan amino acid sequence selected from the group consisting of SEQ IDNOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 29, and 35.For example, the V_(H) regions can comprise an amino acid sequenceselected from the group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 26, 27, 29, and 35.

The invention also provides an anti-PcrV where the V_(L) regionV-segment comprises at least 80% or 90% identity, to a human germlineVkappa I L12 or Vkappa III sequence; or at least 80% or 90% identity toa human germline Vlambda3 31 or to a Vlambda2 2c sequence. In someembodiments, V_(L) region V-segment has at least 80% or 90% identity tothe human germline VKI L12 segment and the CDR1 has the sequenceRASX₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁A (SEQ ID NO:64), where X₁₅ is Q or E; X₁₆ is Sor G; X₁₇ is I or V; X₁₈ is S or D; X₁₉ is S, R, or T; X₂₀ is W or Y;and X₂₁ is L or V; and the CDR2 has the sequence X₂₁ASX₂₂LX₂₃S (SEQ IDNO:65), wherein X₂₁ is D or A; X₂₂ is S, A, or T; and X₂₃ is E, Q, or K.In some embodiments, the CDR1 has the sequence RASQGISTYLA (SEQ IDNO:66), RASQGISSWLA (SEQ ID NO:67), RASQSISRWVA (SEQ ID NO:68), orRASEGVDRWLA (SEQ ID NO:69); or the CDR2 has the sequence AASSLQS (SEQ IDNO:70), DASSLKS (SEQ ID NO:71), DASALQS (SEQ ID NO:72), or DASTLQS (SEQID NO:73). In some embodiments, the CDR1 has the sequence RASQGISTYLA(SEQ ID NO:66), RASQGISSWLA (SEQ ID NO:67), RASQSISRWVA (SEQ ID NO:68),or RASEGVDRWLA (SEQ ID NO:69); and the CDR2 has the sequence AASSLQS(SEQ ID NO:70), DASSLKS (SEQ ID NO:71), DASALQS (SEQ ID NO:72), orDASTLQS (SEQ ID NO:73).

The invention also provides an anti-PcrV antibody where the V_(L) regionV segment has at least 80%, or at least 90%, amino acid sequenceidentity to the human germline VKIII L2 sequence and the CDR1 has thesequence RASNSVGAYNLA (SEQ ID NO:74) or RASQSVSSNLA (SEQ ID NO:75); orthe CDR2 has the sequence (A/G)AS(T/R)RA(T/P) (SEQ ID NO:76). In someembodiments, CDR1 has the sequence RASNSVGAYNLA (SEQ ID NO:74) orRASQSVSSNLA (SEQ ID NO:75); and the CDR2 has the sequence(A/G)AS(T/R)RA(T/P) (SEQ ID NO:76).

Further, the invention provides an anti-PcrV antibody where the V_(L)region V-segment has at least 80%, or at least 90%, amino acid sequenceidentity to a human germline Vlambda L3 31 segment and the CDR1 has thesequence QGDSLRS(Y/L)YAS (SEQ ID NO:77); or the CDR2 has the sequence(G/S)KN(N/S)RPS (SEQ ID NO:78). In some embodiments, the CDR1 has thesequence QGDSLRS(Y/L)YAS (SEQ ID NO:77); and the CDR2 has the sequence(G/S)KN(N/S)RPS (SEQ ID NO:78).

In some embodiments, the invention provide an anti-PcrV antibody wherethe V_(L) region V-segment has at least 80%, or at least 90%, amino acidsequence identity to a human germline Vlambda L2 2c segment and the CDR1has the sequence TGTSSDVGAYNYVS (SEQ ID NO:79) or TGTSSDYVS (SEQ IDNO:80); or the CDR2 has the sequence (E/D)VT(K/N)RPS (SEQ ID NO:81). Insome embodiments, the CDR1 has the sequence TGTSSDVGAYNYVS (SEQ IDNO:79) or TGTSSDYVS (SEQ ID NO:80); and the CDR2 has the sequence(E/D)VT(K/N)RPS (SEQ ID NO:81).

An anti-PcrV antibody of the invention can have a region that comprisesthe V-segment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28,30, 32, 34, 36, and 37. For example the V_(L) region can comprise anamino acid sequence selected from the group consisting of SEQ ID NO:2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 37.

The invention thus provides an anti-PcrV antibody that comprises: aV_(H) region having an amino acid sequence selected from the groupconsisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,26, 27, 29, and 35; and a V_(L) region having an amino acid sequenceselected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 37. Thus, in someembodiments, an antibody of the invention comprises a V_(H) region ofSEQ ID NO:1 and a V_(L) region of SEQ ID NO:2; or a V_(H) region of SEQID NO:3 and a V_(L) region of SEQ ID NO:4; or a V_(H) region of SEQ IDNO:5 and a V_(L) region of SEQ ID NO:6; or a V_(H) region of SEQ ID NO:7and a V_(L) region of SEQ ID NO:8; or a V_(H) region of SEQ ID NO:11 anda V_(L) region of SEQ ID NO:12; or a V_(H) region of SEQ ID NO:9 and aV_(L) region of SEQ ID NO:10; or a V_(H) region of SEQ ID NO:13 and aV_(L) region of SEQ ID NO:10; or a V_(H) region of SEQ ID NO:13 and aV_(L) region of SEQ ID NO:4; or a V_(H) region of SEQ ID NO:13 and aV_(L) region of SEQ ID NO:37; or a V_(H) region of SEQ ID NO:21 and aV_(L) region of SEQ ID NO:18; or a V_(H) region of SEQ ID NO:17 and aV_(L) region of SEQ ID NO:18; or a V_(H) region of SEQ ID NO:26 and aV_(L) region of SEQ ID NO:24; or a V_(H) region of SEQ ID NO:25 and aV_(L) region of SEQ ID NO:24; or a V_(H) region of SEQ ID NO:23 and aV_(L) region of SEQ ID NO:24; or a V_(H) region of SEQ ID NO:35 and aV_(L) region of SEQ ID NO:36; or V_(H) region of SEQ ID NO:29 and aV_(L) region of SEQ ID NO:20; or V_(H) region of SEQ ID NO:29 and aV_(L) region of SEQ ID NO:28; or a V_(H) region of SEQ ID NO:29 and aV_(L) region of SEQ ID NO:30; or a V_(H) region of SEQ ID NO:29 and aV_(L) region of SEQ ID NO:34; or a V_(H) region of SEQ ID NO:3 and aV_(L) region of SEQ ID NO:32.

In some embodiments, an anti-PcrV antibody of the invention comprises aheavy chain as set forth in FIG. 1 and/or a light chain as set forth inFIG. 2; or has at least one, often at least two, and in someembodiments, at least three CDRs from one of the heavy or light chainsset forth in FIG. 1 or FIG. 2, respectively. In many embodiments, theCDR1 and/or CDR2 sequence is not a germline sequence.

In some embodiments, the antibody is a Fab′ fragment.

In some embodiments, an antibody of the invention is a Fab, or Fab′,that has an affinity of about 10 nM or less. In some embodiments, theantibody has an affinity that is equal or better than, the affinity of aMab166 Fab or Fab′.

The potency of an antibody of the invention, e.g., a Fab, in inhibitingthe activity of the P. aeruginosa Type III Secretion System is typicallyequivalent to Mab 166 Fab (within two-fold of the activity in cell-basedassays). In some embodiments, an antibody of the invention is morepotent than Mab 166 in preventing cytotoxicity by P. aeruginosa.

In some embodiments, the antibody comprises a hinge region.

In other embodiments, the antibody is an IgG or an IgA.

In some embodiments, the antibody is PEGylated. For example, theantibody can be di-PEGylated.

In some embodiments, the V_(H) region or the V_(L) region, or both theV_(H) and V_(L) region amino acid sequences comprise a methionine at theN-terminus.

In a further aspect, the invention provides a method of treating apatient infected with P. aeruginosa, the method comprising administeringa therapeutically effective amount of an antibody as described herein.In some embodiments, the antibody is administered intravenously,subcutaneously, or by insufflation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows sequences of V_(H) regions of anti-PcrV antibodies (SEQ IDNOS:1, 7, 9, 5, 11, 3, 19, 19, 19, 13, 15, 17, 21, 23, 25, 26 and 35,respectively). CDR sequences are underlined. The VH1 sequence is alignedto human germ-line sequence VH1-18 (SEQ ID NO:82). VH3-subclassantibodies are shown aligned to human germ-line sequence VH3-30.3 (SEQID NO:83). J-segments are aligned to either human germ-line JH3 or JH6(SEQ ID NO:84). The V_(H)-segments depicted in FIG. 1 correspond to thesequence up to the CDR3 sequence.

FIG. 2 shows sequences of V_(L) regions of anti-PcrV antibodies (SEQ IDNOS:24, 2, 8, 10, 10, 6, 12, 4, 37, 36, 87, 18, 20, 18, 30, 32, 90 and28, respectively). CDR sequences are underlined. Vkappa-subclassantibodies are shown aligned to human germline sequence VkI L12 (SEQ IDNO:85) or VkIII L2 (SEQ ID NO:86). J-segments are aligned to humangerm-line Jk2. Vlambda-subclass antibodies are shown aligned to humangermline sequence VL3 31 (SEQ ID NO:88) or VL2 2c (SEQ ID NO:89).J-segments are aligned to human germ-line J12.

FIG. 3 shows an exemplary constant region sequence that can be linked toFab′ Heavy (SEQ ID NO:91) and Light (SEQ ID NO:92) Chains. The twocysteine residues in the hinge region available for conjugation ofthiol-reactive maleimide derivatives are underlined. The cysteineresidues involved in formation of an interchain disulfide bond areboxed.

FIG. 4 shows a schematic representation of the structure of adi-PEGylated Fab′ in which two molecules of mPEG-maleimide areconjugated via thioether linkages to cysteine residues in the hingeregion of the Fab′ protein. The exemplary antibody is comprised of ahuman Fd′ heavy chain and human kappa light chain linked by a disulphidebond represented by the cross-bar between the two chains. (Fab and PEGcomponents are not to scale.)

FIG. 5 provides data showing a time course of survival of mice treatedwith various doses of antibodies to PcrV at the time of challenge with alethal dose of PA103. Mab166 and Fab fragments were co-instilled via theintratracheal route with 1.5×106 bacteria (5 mice per group, 4 mice forMab166 Fab groups). Control is a nonspecific Fab with no binding to PcrVor any P. aeruginosa protein. Mice were treated with antibody doses of:A) 10 μg, B) 5 μg, C) 2.5 μg, D) 1.25 μg, *P=0.01 for Fab 1A8 vs. Mab166Fab E) 0.625 μg *P=0.002 for 1A8 vs. Mab166 Fab, F) 0.3125 μg, G) 0.16μg, H) 0.08 μg. P values for differences between treatment groupsdetermined by Mantel-Cox log-rank test.

FIG. 6 provides data showing a body temperature analysis of mice treatedwith anti-PcrV antibodies. Rectal temperatures are shown for 48 hours oruntil mortality. Antibody doses: A) 10 μg, B) 5 μg, C) 2.5 μg, D) 1.25μg, E) 0.625 μg F) 0.3125 μg, G) 0.16 μg, H) 0.08 μg.

FIG. 7 provides data showing clearance of P. aeruginosa from the lungsof infected mice by anti-PcrV antibodies. Mice were infected with1.5×10⁶ cfu PA103 co-instilled with Mab 166 IgG, Mab 166 Fab or humanFab 1A8 at the doses shown (in μg). The graph shows cfu/lung isolatedfrom individual mice surviving at 48 h. The number of dead mice at thistime point is shown above the figure. Median cfu/lung for surviving micein each group is shown with a bar.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, an “antibody” refers to a protein functionally definedas a binding protein and structurally defined as comprising an aminoacid sequence that is recognized by one of skill as being derived fromthe framework region of an immunoglobulin-encoding gene of an animalthat produces antibodies. An antibody can consist of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains, respectively.

The term “antibody” as used herein includes antibody fragments thatretain binding specificity. For example, there are a number of wellcharacterized antibody fragments. Thus, for example, pepsin digests anantibody below the disulfide linkages in the hinge region to produceF(ab)′₂, a dimer of Fab which itself is a light chain joined to VH-CH1(Fd) by a disulfide bond. The F(ab)′₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the (Fab′)₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially a Fab with all or part of the hinge region (see, FundamentalImmunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a moredetailed description of other antibody fragments). While variousantibody fragments are defined in terms of the digestion of an intactantibody, one of skill will appreciate that fragments can be synthesizedde novo either chemically or by utilizing recombinant DNA methodology.Thus, the term antibody, as used herein also includes antibody fragmentseither produced by the modification of whole antibodies or synthesizedusing recombinant DNA methodologies.

Antibodies of the invention include dimers such as V_(H)-V_(L) dimers,V_(H) dimers, or V_(L) dimers, including single chain antibodies(antibodies that exist as a single polypeptide chain), such as singlechain Fv antibodies (sFv or scFv) in which a variable heavy and avariable light region are joined together (directly or through a peptidelinker) to form a continuous polypeptide. The single chain Fv antibodyis a covalently linked V_(H)-V_(L) heterodimer which may be expressedfrom a nucleic acid including V_(H)- and V_(L)-encoding sequences eitherjoined directly or joined by a peptide-encoding linker (e.g., Huston, etal. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). While the V_(H) andV_(L) are connected to each as a single polypeptide chain, the V_(H) andV_(L) domains associate non-covalently. Alternatively, the antibody canbe another fragment, such as a disulfide-stabilized Fv (dsFv). Otherfragments can also be generated, including using recombinant techniques.The scFv antibodies and a number of other structures converting thenaturally aggregated, but chemically separated light and heavypolypeptide chains from an antibody V region into a molecule that foldsinto a three dimensional structure substantially similar to thestructure of an antigen-binding site are known to those of skill in theart (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778). Insome embodiments, antibodies include those that have been displayed onphage or generated by recombinant technology using vectors where thechains are secreted as soluble proteins, e.g., scFv, Fv, Fab, (Fab′)₂ orgenerated by recombinant technology using vectors where the chains aresecreted as soluble proteins. Antibodies for use in the invention canalso include diantibodies and miniantibodies.

Antibodies of the invention also include heavy chain dimers, such asantibodies from camelids. Since the V_(H) region of a heavy chain dimerIgG in a camelid does not have to make hydrophobic interactions with alight chain, the region in the heavy chain that normally contacts alight chain is changed to hydrophilic amino acid residues in a camelid.V_(H) domains of heavy-chain dimer IgGs are called VHH domains.Antibodies of the invention include single domain antibodies (dAbs) andnanobodies (see, e.g., Cortez-Retamozo, et al., Cancer Res.64:2853-2857, 2004).

As used herein, “V-region” refers to an antibody variable region domaincomprising the segments of Framework 1, CDR1, Framework 2, CDR2, andFramework 3, including CDR3 and Framework 4, which segments are added tothe V-segment as a consequence of rearrangement of the heavy chain andlight chain V-region genes during B-cell differentiation. A “V-segment”as used herein refers to the region of the V-region (heavy or lightchain) that is encoded by a V gene. The V-segment of the heavy chainvariable region encodes FR1-CDR1-FR2-CDR2 and FR3. For the purposes ofthis invention, the V-segment of the light chain variable region isdefined as extending though FR3 up to CDR3.

As used herein, the term “J-segment” refers to a subsequence of thevariable region encoded comprising a C-terminal portion of a CDR3 andthe FR4. An endogenous J-segment is encoded by an immunoglobulin J-gene.

As used herein, “complementarity-determining region (CDR)” refers to thethree hypervariable regions in each chain that interrupt the four“framework” regions established by the light and heavy chain variableregions. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, for example, a V_(H) CDR3 is located in the variabledomain of the heavy chain of the antibody in which it is found, whereasa V_(L) CDR1 is the CDR1 from the variable domain of the light chain ofthe antibody in which it is found.

The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The amino acid sequences of the CDRs and framework regions can bedetermined using various well known definitions in the art, e.g., Kabat,Chothia, international ImMunoGeneTics database (IMGT), and AbM (see,e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structuresfor the hypervariable regions of immunoglobulins. J. Mol. Biol. 196,901-917; Chothia C. et al., 1989, Conformations of immunoglobulinhypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992,structural repertoire of the human VH segments J. Mol. Biol. 227,799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). Definitions ofantigen combining sites are also described in the following: Ruiz etal., IMGT, the international ImMunoGeneTics database. Nucleic AcidsRes., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the internationalImMunoGeneTics database. Nucleic Acids Res. January 1; 29(1):207-9(2001); MacCallum et al, Antibody-antigen interactions: Contact analysisand binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); andMartin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin,et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al,Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M. J. E.(ed.), Protein Structure Prediction. Oxford University Press, Oxford,141-172 1996).

“Epitope” or “antigenic determinant” refers to a site on an antigen towhich an antibody binds. Epitopes can be formed both from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996).

The term “binding specificity determinant” or “BSD” as used in thecontext of the current invention refers to the minimum contiguous ornon-contiguous amino acid sequence within a CDR region necessary fordetermining the binding specificity of an antibody. In the currentinvention, the minimum binding specificity determinants reside within aportion or the full-length of the CDR3 sequences of the heavy and lightchains of the antibody.

As used herein, the terms “PcrV antagonizing antibody” or an “anti-PcrVantibody is an antagonist of the Pseudomonas aeruginosa Type IIIsecretion system” are used interchangeably to refer to an antibody thatbinds to PcrV and inhibits the Type III secretion system. Inhibitionoccurs when secretion through the Type III secretion system is at leastabout 10% less, for example, at least about 25%, 50%, 75% less, ortotally inhibited, in comparison to secretion when not exposed to theantibody antagonist. The terms “anti-PcrV antibody” and “PcrV antibody”are used synonymously unless otherewise stated.

The term “equilibrium dissociation constant (K_(D)) refers to thedissociation rate constant (k_(d), time⁻¹) divided by the associationrate constant (k_(a), time⁻¹, M⁻¹). Equilibrium dissociation constantscan be measured using any known method in the art. The antibodies of thepresent invention are high affinity antibodies. Such antibodies have anaffinity better than 500 nM, and often better than 50 nM or 10 nM. Thus,in some embodiments, the antibodies of the invention have an affinity inthe range of 500 nM to 100 pM, or in the range of 50 or 25 nM to 100 pM,or in the range of 50 or 25 nM to 50 pM, or in the range of 50 nM or 25nM to 1 pM.

As used herein, “humanized antibody” refers to an immunoglobulinmolecule in CDRs from a donor antibody are grafted onto human frameworksequences. Humanized antibodies may also comprise residues of donororigin in the framework sequences. The humanized antibody can alsocomprise at least a portion of a human immunoglobulin constant region.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. Humanization can be performed using methods known in the art(e.g., Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988);Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; U.S. Pat. No.4,816,567), including techniques such as “superhumanizing” antibodies(Tan et al., J. Immunol. 169: 1119, 2002) and “resurfacing” (e.g.,Staelens et al., Mol. Immunol. 43: 1243, 2006; and Roguska et al., Proc.Natl. Acad. Sci USA 91: 969, 1994).

A “humaneered” antibody in the context of this invention refers to anengineered human antibody having a binding specificity of a referenceantibody. A “humaneered” antibody for use in this invention has animmunoglobulin molecule that contains minimal sequence derived from adonor immunoglobulin. Typically, an antibody is “humaneered” by joininga DNA sequence encoding a binding specificity determinant (BSD) from theCDR3 region of the heavy chain of the reference antibody to human V_(H)segment sequence and a light chain CDR3 BSD from the reference antibodyto a human V_(L) segment sequence. A “BSD” refers to a CDR3-FR4 region,or a portion of this region that mediates binding specificity. A bindingspecificity determinant therefore can be a CDR3-FR4, a CDR3, a minimalessential binding specificity determinant of a CDR3 (which refers to anyregion smaller than the CDR3 that confers binding specificity whenpresent in the V region of an antibody), the D segment (with regard to aheavy chain region), or other regions of CDR3-FR4 that confer thebinding specificity of a reference antibody. Methods for humaneering areprovided in US patent application publication no. 20050255552 and USpatent application publication no. 20060134098.

The term “hybrid” when used with reference to portions of a nucleic acidor protein, indicates that the nucleic acid or protein comprises two ormore subsequences that are not normally found in the same relationshipto each other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid.Similarly, a hybrid protein refers to two or more subsequences that arenot normally found in the same relationship to each other in nature.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as depicted above.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction where the antibodybinds to the protein of interest. In the context of this invention, theantibody typically binds to PcrV with an affinity of 500 nM or less, andhas an affinity of 5000 nM or greater, for other antigens.

The terms “identical” or percent “identity,” in the context of two ormore polypeptide (or nucleic acid) sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues (or nucleotides) that are the same(i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site). Such sequences are then said to be “substantiallyidentical.” “Substantially identical” sequences also includes sequencesthat have deletions and/or additions, as well as those that havesubstitutions, as well as naturally occurring, e.g., polymorphic orallelic variants, and man-made variants. As described below, thepreferred algorithms can account for gaps and the like. Preferably,protein sequence identity exists over a region that is at least about 25amino acids in length, or more preferably over a region that is 50-100amino acids in length, or over the length of a protein.

A “comparison window”, as used herein, includes reference to a segmentof one of the number of contiguous positions selected from the groupconsisting typically of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

Preferred examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity include the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990). BLAST and BLAST 2.0 are used, with the parameters describedherein, to determine percent sequence identity for the nucleic acids andproteins of the invention. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. The term“purified” in some embodiments denotes that a protein gives rise toessentially one band in an electrophoretic gel. Preferably, it meansthat the protein is at least 85% pure, more preferably at least 95%pure, and most preferably at least 99% pure.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine Thus, at every position where an alanine is specifiedby a codon, the codon can be altered to another of the correspondingcodons described without altering the encoded polypeptide. Such nucleicacid variations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes silent variations of thenucleic acid. One of skill will recognize that in certain contexts eachcodon in a nucleic acid (except AUG, which is ordinarily the only codonfor methionine, and TGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Accordingly, often silent variations of a nucleic acid which encodes apolypeptide is implicit in a described sequence with respect to theexpression product, but not with respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables and substitution matrices such asBLOSUM providing functionally similar amino acids are well known in theart. Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. Typical conservative substitutions for one anotherinclude: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

I. Introduction

The invention relates to antibodies that bind with high affinity to thePcrV antigen from Pseudomonas aeruginosa and are typically functionalantagonists of the Type III secretion system. The antibodies comprisevariable regions with a high degree of homology to human germ-line V_(H)and V_(L) sequences. The CDR3 sequences of the heavy and light chainscomprise a pair of binding specificity determinants (BSD) from themonoclonal anti-PcrV antibody Mab166 (Frank et al., J. Infectious Dis.186: 64-73, 2002; and U.S. Pat. No. 6,827,935) and the antibodies of theinvention compete with Mab166 for binding to a neutralizing epitope onthe PcrV protein (see, e.g., U.S. Pat. No. 6,827,935).

The BSD sequence in CDRH3 has the amino acid sequence NRGDIYYDFTY (SEQID NO:38. In some embodiments, an antibody of the invention as a heavychain CDR3 sequence NRGDIYYDFTYA(M/F)DX (SEQ ID NO:93), where X is I, S,or Q.

The BSD in CDRL3 is FWXTP (where X may be either S or G; SEQ ID NO:39).Complete V-regions are generated in which the BSD forms part of the CDR3and additional sequences are used to complete the CDR3 and add a FR4sequence. Typically, the portion of the CDR3 excluding the BSD and thecomplete FR4 are comprised of human germ-line sequences. In preferredembodiments, the CDR3-FR4 sequence excluding the BSD differs from humangerm-line sequences by not more than 2 amino acids on each chain.

The human germline V-segment repertoire consists of 51 heavy chainV-segments, 40 κ light chain V-segments, and 31 λ light chainV-segments, making a total of 3,621 germline V-region pairs. Inaddition, there are stable allelic variants for most of theseV-segments, but the contribution of these variants to the structuraldiversity of the germline repertoire is limited. The sequences of allhuman germ-line V-segment genes are known and can be accessed in theV-base database (on the worldwide web at vbase.mrc-cpe.cam.ac.uk),provided by the MRC Centre for Protein Engineering, Cambridge, UnitedKingdom (see, also Chothia et al., 1992, J Mol Biol 227:776-798;Tomlinson et al., 1995, EMBO J 14:4628-4638; Cook et al. (1995) Immunol.Today 16: 237-242and Williams et al., 1996, J Mol Biol 264:220-232); orthe international ImMunoGeneTics database (IMGT). These sequences can beused as reference sources for the human germline segments of theantibodies of the invention.

Antibodies or antibodies fragments as described herein can be expressedin prokaryotic or eukaryotic microbial systems or in the cells of highereukaryotes such as mammalian cells.

An antibody that is employed in the invention can be in any format. Forexample, in some embodiments, the antibody can be a complete antibodyincluding a constant region, e.g., a human constant region, or can be afragment or derivative of a complete antibody, e.g., a Fab, Fab′,F(ab′)₂, scFv, Fv, or a single domain antibody, such as a nanobody or acamelid antibody.

II. Heavy Chains

A heavy chain of an anti-PcrV antibody of the invention comprises aheavy-chain V-region that comprises the following elements:

1) human heavy-chain V-segment sequences comprisingFR1-CDR1-FR2-CDR2-FR3

2) a CDRH3 region comprising the amino acid sequence NRGDIYYDFTY (SEQ IDNO:33)

3) a FR4 contributed by a human germ-line J-gene segment.

Examples of V-segment sequences that support binding to PcrV incombination with a CDR3-FR4 segment described above together with acomplementary V_(L) region are shown in FIG. 1. The V-segments can befrom the human VH1 or VH3 sub-classes. In some embodiments, theV-segment is a human V_(H)3 sub-class segment that has a high degree ofamino-acid sequence identity with the germ-line segment VH3-30.3. Forexample the V-segment differs by not more than fifteen residues fromVH3-30.3 and preferably not more than seven residues.

The FR4 sequence of the antibodies of the invention is provided by ahuman J segment. There are six heavy chain JH-regions numbered 1 through6. Thus, the FR4 sequences can be provided by a JH1, JH2, JH3, JH4, JH5or JH6 gene segment. Typically, the FR4 region of an antibody of theinvention has at least 90%, often at least 91%, 92%, 93%, 94%, 95% 96%,97%, 98%, 99%, or 100% identity, to the FR4 region of the human germlineJ segment that provides the FR4.

In some embodiments, the FR4 sequence is provided by a human germ-lineJH3 segment and has a sequence WGQGTMVTVSS (SEQ ID NO:94). In otherembodiments, the FR4 is provided by a human germ-line JH6 segment andhas the sequence WGQGTTVTVSS (SEQ ID NO:95).

The CDRH3 also comprises sequences that are derived from a humanJ-segment. Typically, the CDRH3-FR4 sequence excluding the BSD differsby not more than 2 amino acids from a human germ-line J-segment. Intypical embodiments, the J-segment sequences in CDRH3 are from the sameJ-segment used for the FR4 sequences. Thus, in some embodiments, theCDRH3-FR4 region comprises the BSD and a complete human JH3 germ-linegene segment. Exemplary combinations of CDRH3 and FR4 sequences areshown below, in which the BSD is in bold and human germ-line J-segmentresidues are underlined:

(SEQ ID NO: 96)      CDR3 NRGDIYYDFTY AFDI WGQGTMVTVSS(FR4 = JH3) (SEQ ID NO: 97) NRGDIYYDFTY AMDIWGQGTMVTVSS(FR4 = JH3)  (SEQ ID NO: 98)NRGDIYYDFTYAMDIWGQGTTVTVSS(FR4 = JH6) 

In some embodiments, an antibody of the invention comprises a V-segmentthat has at least 90% identity, or at least 91%, 92% 93%, 94%, 95%, 965,97%, 98%, 99%, or 100% identity to the germ-line segment VH3 30.3 or toa germlineVH1-18 segment; or to one of the V-segments of the V_(H)regions shown in FIG. 1, such as a V-segment portion of SEQ ID NOS:1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 29, and 35.

In some embodiments, the V-segment of the VH region has a CDR1 and/orCDR2 as shown in FIG. 1. For example, an antibody of the invention mayhave a CDR1 that has the sequence TAGMH (SEQ ID NO:50), SYGIH (SEQ IDNO:51), SYGMH (SEQ ID NO:52), SYPLH (SEQ ID NO:53), or NYPMH (SEQ IDNO:54); or a CDR2 that has the sequence VIWYNGKEISYADSVKG (SEQ IDNO:55), FISYDGSEKYYASSVKG (SEQ ID NO:56), or VISYDGSEKWYADSVKG (SEQ IDNO:57). In some embodiments, the CDR2 of the VH region has a negativelycharged amino acid positioned in about the middle, e.g., at position 8or 9 of the CDR2.

In particular embodiments, an antibody has both a CDR1 and a CDR2 fromone of the V_(H) region V-segments shown in FIG. 1 and a CDR3 thatcomprises NRGDIYYDFTY (SEQ ID NO:38), e.g., NRGDIYYDFTYAFDI (SEQ IDNO:63) or NRGDIYYDFTYAMDI (SEQ ID NO:99). Thus, an anti-PcrV antibody ofthe invention, may for example, have a CDR3-FR4 that has the sequenceNRGDIYYDFTYAFDIWGQGTMVTVSS (SEQ ID NO:96), NRGDIYYDFTYAMDIWGQGTMVTVSS(SEQ ID NO:97), or NRGDIYYDFTYAMDIWGQGTTVTVSS (SEQ ID NO:98). In otherembodiments, the antibody may comprise a CDR3 that has the sequenceNRGDIYYDFTYA(M/F)D(Q/S) (SEQ ID NO:100).

III. Light Chains

A light chain of an anti-PcrV antibody of the invention comprises atlight-chain V-region that comprises the following elements:

1) human light-chain V-segment sequences comprisingFR1-CDR1-FR2-CDR2-FR3

2) a CDRL3 region comprising the sequence FWXTP (where X may be S or G;SEQ ID NO:39)

3) a FR4 contributed by a human germ-line J-gene segment.

The V_(L) region comprises either a Vlambda or a Vkappa V-segment.Examples of Vlambda and Vkappa sequences that support binding incombination with a complementary V_(H)-region are provided in FIG. 2.Vkappa segments are cloned upstream of the human germ-line JK2 segmentand Vlambda segments are cloned upstream of the germ-line JL2 segment.

The CDRL3 sequence comprises a V-segment and J-segment derivedsequences. In typical embodiments, the J-segment sequences in CDRL3 arefrom the same J-segment used for FR4. Thus, may differ by not more than2 amino acids from human kappa germ-line V-segment and J-segmentsequences. In some embodiments, the CDRL3-FR4 region comprises the BSDand the complete human JK2 germ-line gene segment. Exemplary CDRL3-FR4combinations for kappa chains are shown below in which the BSD is shownin bold and JK2 sequences are underlined:

  CDR3 QQ FWSTP YT FGQGTKLEIK (JK2)(SEQ ID NO: 101) QHFWGTPYTFGQGTKLEIK (JK2)(SEQ ID NO: 102)

A preferred CDR3-FR4 for lambda chains is shown below in which the BSDis shown in bold and the JL2 sequences are underlined:

  CDR3 QH FWSTP YT F GGGTKLTVL (JL2)(SEQ ID NO: 103)

The FR4 sequence of the antibodies of the invention is provided by ahuman J segment. There are five human JKappa-region segments labeled 1though 5 and four JLambda-region segments labeled 1, 2, 3 and 7. Thus,the FR4 sequences can be provided by by any of these germline sequences.Typically, the FR4 region of an antibody of the invention has at least90%, often at least 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100%identity, to the FR4 region of the human germline J segment thatprovides the FR4.

The Vkappa segments are typically of the VKI or VKIII sub-class. In someembodiments, the segments have at least 80% sequence identity to a humangermline VKI or VKIII subclass, e.g., at least 80% identity to the humangerm-line VKI L12 sequence or to human germline VKIII L2 or VKIIIAl lsequence. For example, the Vkappa segment may differ by not more than 18residues from VKI L12, or 12 residues from VKIII A11 or VKIII L2. Inother embodiments, the V_(L) region V-segment of an antibody of theinvention has at least 85% identity, or at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the human germline VKIL12, or to the human germline VkIII L2 sequence, or to human germlineVKIII A11 sequence, or to a kappa V-segment sequence of a V_(L) regionshown in FIG. 2, for example, the V-segment sequence of SEQ ID NOS:2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 37.

In some embodiments, the V-segment of the V_(L) corresponds to a humangermline Vlambda segment. Thus, in some embodiments, the V-segment hasat least 85% identity, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to a Vlambda V-segment of a V_(L) regionof FIG. 2, such as the V-segment sequence of SEQ ID NOS:28, 30, 32, or34.

In some embodiments, the V-segment of the V_(L) region has a CDR1 and/orCDR2 as shown in FIG. 2. For example, an antibody of the invention mayhave a CDR1 sequence of RASQGISTYLA (SEQ ID NO:66), RASQGISSWLA (SEQ IDNO:67), RASQSISRWVA (SEQ ID NO:68), or RASEGVDRWLA (SEQ ID NO:69) orCDR2 sequence AASSLQS (SEQ ID NO:70), DASSLKS (SEQ ID NO:71), DASALQS(SEQ ID NO:72), or DASTLQS (SEQ ID NO:73). In other embodiments, theantibody may have a CDR1 sequence of QGDSLRSYYA (SEQ ID NO:104),TGTSSDVGAYNYVS (SEQ ID NO:79), or TGTSSDYV (SEQ ID NO:105); or a CDR2sequence GKNNRPS (SEQ ID NO:106), EVTKRPS (SEQ ID NO:107), or DVTNRPS(SEQ ID NO:108).

In particular embodiments, an anti-PcrV antibody of the invention mayhave a CDR1 and a CDR2 in a combination as shown in one of theV-segments of the V_(L) regions set forth in FIG. 2 and a CDR3 sequencethat comprises FWXTP (SEQ ID NO:39), where X is S or G, e.g., the CDR3may be QQFWSTPYT (SEQ ID NO:109), QHFWGTPYT (SEQ ID NO:110), orQHFWSTPYT (SEQ ID NO:111). In some embodiments, such an anti-PcrVantibody may comprise an FR4 region that is FGQGTKLEIK (SEQ ID NO:47) orFGGGTKLTVL (SEQ ID NO:48). Thus, an anti-PcrV antibody of the invention,can comprise, e.g., both the CDR1 and CDR2 from one of the V_(L) regionsshown in FIG. 2 and a CDR3-FR4 region that is QQFWSTPYTFGQGTKLEIK (SEQID NO:101), QHFWGTPYTFGQGTKLEIK (SEQ ID NO:102), or QHFWSTPYTFGGGTKLTVL(SEQ ID NO:103).

IV. Preparation of PcrV Antibodies

An antibody of the invention may comprise any of the V_(H) regions ofSEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 29, or35 in combination with any of the V_(L) regions of SEQ ID NOS:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36 or 37.

An antibody may be tested to confirm that the antibody retains theactivity of antagonizing the Type III secretion system. The antagonistactivity can be determined using any number of endpoints, includingcytotoxicity assays. Exemplary assays are described, e.g., in U.S. Pat.No. 6,827,935. An antibody that is administered to treat P. aeruginosainfection preferably retains at least 75%, preferably 80%, 90%, 95%, or100%, of the Type III secretion pathway antagonist activity of Mab166(U.S. Pat. No. 6,827,935).

A high-affinity antibody may be identified using well known assays todetermine binding activity and affinity. Such techniques include ELISAassays as well as binding determinations that employ surface plasmonresonance or interferometry. For example, affinities can be determinedby biolayer interferometry using a ForteBio (Mountain View, Calif.)Octet biosensor.

Antibodies of the invention typically compete with Mab166 for binding toPcrV. The region of PcrV to which Mab166 binds has been identified (U.S.Pat. No. 6,827,935). PcrV or a fragment thereof that binds Mab166 can beemployed in a competitive binding assay. The ability of an antibodydescribed herein to block or compete with Mab166 for binding to PcrVindicates that the antibody binds to the same epitope as Mab166 or to anepitope that is close to, e.g., overlapping, with the epitope that isbound by Mab166. In other embodiments an antibody described herein,e.g., an antibody comprising a V_(H) and V_(L) region combination asshown in Table 1, can be used as a reference antibody for assessingwhether another antibody competes for binding to PcrV. A test antibodyis considered to competitively inhibit binding of a reference antibody,if binding of the reference antibody to the antigen is reduced by atleast 30%, usually at least about 40%, 50%, 60% or 75%, and often by atleast about 90%, in the presence of the test antibody. Many assays canbe employed to assess binding, including ELISA, as well as other assays,such as immunoblots.

In some embodiments, the anti-PcrV antibody need not antagonize the TypeIII secretion sequences. For example, antibodies of the invention thatbind to PcrV can recruit multiple cell types of the immune system tostimulate phagocytosis by macrophages, antibody directed cellularcytotoxicity (ADCC) by macrophages or NK cells, activation of thecomplement cascade, and/or generation of the oxidative burst byneutrophils, thereby causing bacterial, i.e., P. aeruginosa, death.Furthermore, all antibody variable regions are capable of catalyzingredox reactions from singlet oxygen provided by activated neutrophils,leading to the generation of a variety of highly potent oxidizing agentsdirectly harmful to bacteria (see, e.g., Wentworth et al., Proc. Natl.Acad. Sci USA 97:10930-10935, 2000), including ozone, a potentantibacterial agent which also stimulates inflammatory responses (see,e.g., Babior et al., Proc. Natl. Acad. Sci USA 100:3031-3034, 2003).Indeed, inflammation induced by complement activation and ozonegeneration has the potential to recruit additional elements of theimmune system to further boost immunity. Such antibodies typically havean affinity of 50 nM or less, typically less than about 10 nM.

Non-neutralizing and neutralizing anti-PcrV antibodies used incombination with antibiotics provide a strong therapeutic effect.

Methods for the isolation of antibodies with V-region sequences close tohuman germ-line sequences have previously been described (US patentapplications 20050255552 and 20060134098). Antibody libraries may beexpressed in a suitable host cell including mammalian cells, yeast cellsor prokaryotic cells. For expression in some cell systems, a signalpeptide can be introduced at the N-terminus to direct secretion to theextracellular medium. Antibodies may be secreted from bacterial cellssuch as E. coli with or without a signal peptide. Methods forsignal-less secretion of antibody fragments from E. coli are describedin US patent application 20070020685.

To generate a PcrV-binding antibody, one of the V_(H)-regions of theinvention is combined with one of the V_(L)-regions of the invention andexpressed in any of a number of formats in a suitable expression system.Thus the antibody may be expressed as a scFv, Fab, Fab′ (containing animmunoglobulin hinge sequence), F(ab′)₂, (formed by di-sulfide bondformation between the hinge sequences of two Fab′ molecules), wholeimmunoglobulin or truncated immunoglobulin or as a fusion protein in aprokaryotic or eukaryotic host cell, either inside the host cell or bysecretion. A methionine residue may optionally be present at theN-terminus, for example, in polypeptides produced in signal-lessexpression systems. Each of the V_(H)-regions described herein may bepaired with each of the V_(L) regions to generate an anti-PcrV antibody.For example, VH3 1080-2F was identified from the library paired with twodifferent lambda light chains (1080-2F and 1080-11E). The kappa chain1069-3F was identified paired with VH3 1069-3F and with VH3 1100-3.Exemplary combinations of heavy and light chains are shown in the Table1.

TABLE 1 Exemplary antibody heavy-chain and light-chain combinations VHVkappa SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO:3 SEQ ID NO: 12 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQID NO: 5 SEQ ID NO: 6 SEQ ID NO: 13 SEQ ID NO: 37 SEQ ID NO: 21 SEQ IDNO: 18 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 26 SEQ ID NO: 24 SEQ IDNO: 25 SEQ ID NO: 24 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 29 SEQ IDNO: 20 SEQ ID NO: 35 SEQ ID NO: 36 Vlambda SEQ ID NO: 29 SEQ ID NO: 28SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 29 SEQ ID NO: 34 SEQ ID NO: 3 SEQID NO: 32

In many embodiments, the antibodies of the invention antagonize the P.aeruginosa type III secretion system and typically exhibit high affinitybinding to PcrV. High affinity binding between an antibody and anantigen exists if the affinity of the antibody is less than 500 or 100nM, for example, less than 50 nM or less than 25 nM, or less than 10 nM,or less than 1 nM, e.g., less than about 100 pM. The antibodies of theinvention typically have an affinity of 50 nM or less, often 10 nM orless, when assayed as Fabs, e.g., using ELISA, surface plasmon resonanceassays, or interferometry. Table 1 provides examples of such antibodies.

In some embodiments, an antibody of the invention is more potent in acellular cyototxicity assay than Mab166.

Antibodies may be produced using any number of expression systems,including both prokaryotic and eukaryotic expression systems. Many suchsystems are widely available from commercial suppliers. In embodimentsin which an antibody comprises both a V_(H) and V_(L) region, the V_(H)and V_(L) regions may be expressed using a single vector, e.g., in adiscistronic expression unit, or under the control of differentpromoters. In other embodiments, the V_(H) and V_(L) region may beexpressed using separate vectors. The antibodies of the invention may beexpressed with or without a methionine at the N-terminus. Thus, a V_(H)or V_(L) region as described herein may optionally comprise a methionineat the N-terminus.

An antibody of the invention may be produced in any number of formats,including as a Fab, a Fab′, a F(ab′)₂, a scFv, or a dAB. An antibody ofthe invention can also include a human constant region. The constantregion of the light chain may be a human kappa or lambda constantregion. The heavy chain constant region is often a gamma chain constantregion, for example, a gamma-1, gamma-2, gamma-3, or gamma-4 constantregion. In other embodiments, the antibody may be an IgA.

In some embodiments, the antibody is “non-immunogenic” when administeredto a human. The term “non-immunogenic” as used here refers to a PcrVantibody of the invention that does not provoke antibody productionagainst the anti-PcrV antibody when administered to a human. Antibodiescan be assessed for immunogenicity using known assays, e.g., anelectrochemiluminescence immunoassay described in example 5. Such assaysdetect the level of antibodies present in a patient, e.g., in a serumsample from the patient, that react with the anti-PcrV antibody that isadministered to the patient. An assay is considered to show that theantibody is non-immunogenic when no detectable antibody to the anti-PcrVantibody is present in the sample, e.g., in comparison to a controlsample from an individual that was not administered the antibody.

V. PEGylation of Antibodies

In some embodiments, e.g., where the antibody is a fragment, theantibody can be conjugated to another molecule, e.g., polyethyleneglycol (PEGylation) or serum albumin, to provide an extended half-lifein vivo. Examples of PEGylation of antibody fragments are provided inKnight et al. Platelets 15:409, 2004 (for abciximab); Pedley et al., Br.J. Cancer 70:1126, 1994 (for an anti-CEA antibody); Chapman et al.,Nature Biotech. 17:780, 1999; and Humphreys, et al., Protein Eng. Des.20: 227, 2007).

In some embodiments, the antibodies of the invention are in the form ofa Fab′ fragment. A full-length light chain is generated by fusion of aV_(L)-region to human kappa or lambda constant region. Either constantregion may be used for any light chain; however, in typical embodiments,a kappa constant region is used in combination with a Vkappa variableregion and a lambda constant region is used with a Vlambda variableregion.

The heavy chain of the Fab′ is a Fd′ fragment generated by fusion of aV_(H)-region of the invention to human heavy chain constant regionsequences, the first constant (CH1) domain and hinge region. The heavychain constant region sequences can be from any of the immunoglobulinclasses, but is often from an IgG, and may be from an IgG1, IgG2, IgG3or IgG4. The Fab′ antibodies of the invention may also be hybridsequences, e.g., a hinge sequence may be from one immunoglobulinsub-class and the CH1 domain may be from a different sub-class. In apreferred embodiment, the heavy chain constant region including the CH1domain and hinge sequence is from human IgG1.

The Fab′ molecule can be PEGylated using known methods. The hinge regionof the heavy chain contains cysteine residues suitable for conjugationto a polyethylene glycol derivative. The hinge sequence may be thecomplete natural hinge region of an immunoglobulin heavy chain or may betruncated by one or more amino-acids. In some embodiments, the hingeregion may be a modified or synthetic sequence. In further embodiments,the hinge is a natural immunoglobulin hinge sequence and contains twocysteine residues.

In some embodiments, Fab′ molecules can be conjugated by site-specificconjugation to maleimide derivatives of methoxy polyethylene glycol(mPEG-mal). The mPEG-mal can have, for example, an average molecularmass of between 10 and 40 kD. The PEG may be branched PEG or linear PEG.In some embodiments, the mPEG-mal is a linear molecule and has anapproximate molecular weight of 30 kD. One or more molecules of mPEG-malis conjugated to each Fab′ molecule. The mPEG molecules are conjugatedvia thioether linkages between the maleimide moiety of mPEG-mal and oneor more of the cysteine residues in the hinge region of the Fab′ heavychain to form the PEGylated Fab′ molecule. The mPEG-mal is conjugated insuitable buffer and under conditions suitable for thioether formationusing methods known in the art for conjugation of maleimide derivativesto thiol-groups on proteins.

The Fab′ may be produced from the expression system in a form in whichthe hinge cysteine groups are in an oxidized form. In this case, theFab′ may be subjected to a reduction step prior to conjugation. Reducingagents suitable for generation of free hinge thiols and methods forselective reduction of hinge cysteines are known in the art and includethe use of dithiothreitol (DTT), beta-mercapto-ethanol,beta-mercapto-ethylamine (MEA) and non-thiol reducing agents such astris(2-carboxyethyl) phosphine. In some embodiments, the reduction iscarried out under conditions such that the hinge cysteines areselectively reduced and PEGylation occurs predominantly at the hinge.Typically, the PEGylated Fab′ comprises two molecules of mPEG due toPEGylation of both cysteine residues in the hinge. In some embodiments,a mutation may be introduced into the hinge region to replace one of thecysteine residues with another amino acid. Derivatization of such amutant with mPEG-mal leads to the generation of mono-PEGylated Fab′.

Examples of suitable sequences of hinges are:

natural human gamma-1 hinge THTCPPCPA (SEQ ID NO: 112) for di-PEGylationMutant Hinge-1 for mono-PEGylation THT A PPCPA (SEQ ID NO: 113)Mutant Hinge-2 for mono-PEGylation THTCPP A PA (SEQ ID NO: 114)

Other methods of PEGylation, for example, where the PEG is notintroduced at a hinge are also known. For example, Humphreys et al.,supra, describe methods for PEGylation of cysteine residues outside thehinge region by disruption of the interchain disulphide bond between theheavy and light chain of a Fab.

Methods for purification of PEGylated Fab′ and separation of the desiredmono- or di-PEGylated Fab′ from unreacted mPEG-maleimide and Fab′molecules containing higher numbers of PEG moieties are known in theart. Such methods include, for example, size-exclusion or ion-exchangechromatography.

VI. Administration of PcrV Antibodies for the Treatment of P. aeruginosaInfections

The invention also provides methods of treating a patient that has, oris at risk of having, a P. aeruginosa infection by administering anantibody of the invention. In some embodiments, such a patient hascystic fibrosis, ventilator-associated pneumonia (VAP),cancer-associated neutropenia, or burns. The methods of the inventioncomprise administering a PcrV antibody as a pharmaceutical compositionto an P. aeruginosa-infected patient in a therapeutically effectiveamount using a dosing regimen suitable for treatment of the disease. Thecomposition can be formulated for use in a variety of drug deliverysystems. One or more physiologically acceptable excipients or carrierscan also be included in the compositions for proper formulation.Suitable formulations for use in the present invention are found inRemington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed. (1985). For a brief review of methods fordrug delivery, see, Langer, Science 249: 1527-1533 (1990).

The PcrV antibody is provided in a solution suitable for injection intothe patient such as a sterile isotonic aqueous solution for injection.The antibody is dissolved or suspended at a suitable concentration in anacceptable carrier. In some embodiments the carrier is aqueous, e.g.,water, saline, phosphate buffered saline, and the like. The compositionsmay contain auxiliary pharmaceutical substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, and the like.

The pharmaceutical compositions of the invention are administered to apatient, e.g., a cystic fibrosis patient having a P. aeruginosainfection, in an amount sufficient to cure or at least partially arrestthe disease or symptoms of the disease and its complications. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.” A therapeutically effective dose is determined by monitoring apatient's response to therapy. Typical benchmarks indicative of atherapeutically effective dose include amelioration of symptoms ofinfection in the patient, or a decrease in the levels of P. aeruginosain the patient. Amounts effective for this use will depend upon theseverity of the disease and the general state of the patient's health,including other factors such as age, weight, gender, administrationroute, etc. Single or multiple administrations of the antibody may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the methods provide a sufficientquantity of PcrV antibody to effectively treat the patient.

The antibody may be administered alone, or in combination with othertherapies to treat the P. aeruginosa infection.

The antibody can be administered by injection or infusion through anysuitable route including but not limited to intravenous, sub-cutaneous,intramuscular or intraperitoneal routes. In some embodiments, theantibody may be administered by insufflation. In an exemplaryembodiment, the antibody may be stored at 10 mg/ml in sterile isotonicaqueous saline solution for injection at 4° C. and is diluted in either100 ml or 200 ml 0.9% sodium chloride for injection prior toadministration to the patient. The antibody is administered byintravenous infusion over the course of 1 hour at a dose of between 0.2and 10 mg/kg. In other embodiments, the antibody is administered byintravenous infusion over a period of between 15 minutes and 2 hours. Instill other embodiments, the administration procedure is viasub-cutaneous bolus injection.

The dose of antibody is chosen in order to provide effective therapy forthe patient and is in the range of less than 0.1 mg/kg body weight to 25mg/kg body weight or in the range 1 mg-2 g per patient. Preferably thedose is in the range 1-10 mg/kg or approximately 50 mg-1000 mg/patient.The dose may be repeated at an appropriate frequency which may be in therange once per day to once every three months, depending on thepharmacokinetics of the antibody (e.g. half-life of the antibody in thecirculation) and the pharmacodynamic response (e.g. the duration of thetherapeutic effect of the antibody). In some embodiments, the in vivohalf-life of between about 7 and about 25 days and antibody dosing isrepeated between once per week and once every 3 months. In otherembodiments, the antibody is administered approximately once per month.

In further embodiments, the antibody is PEGylated. For example, anantibody of the invention may be PEGylated, e.g., using methods asdescribed herein, and administered to a patient infected with P.aeruginosa.

A V_(H) region and/or V_(L) region of the invention may also be used fordiagnostic purposes. For example, the V_(H) or V_(L) region may be usedfor clinical analysis, such as detection of P. aeruginosa in samplesfrom patient that have, or are suspected of having, a P. aeruginosainfection. A V_(H) or V_(L) region of the invention may also be used,e.g., to produce anti-Id antibodies.

EXAMPLES Example 1 Identification of Engineered Human Anti-Pcrv FabMolecules

Epitope-focused engineered human antibody Fab libraries were generatedas described in US patent application 20050255552. V-segment sequencesderived from repertoires of human immunoglobulin sequences were clonedupstream of a selected CDR3-FR4 sequence for each of the heavy and lightchains.

For heavy-chain repertoires, the CDRH3 comprises a D-segment derivedsequence (NRGDIYYDFTY; SEQ ID NO:38) from a previously identifiedanti-PcrV monoclonal antibody (Mab 166; Frank et at 2002 J. InfectiousDis. 186: 64-73) which constitutes a binding specificity determinant.The sequence of the complete CDRH3-FR4 sequence for the heavy chainrepertoires is shown below.

For VH1 library 1015, the CDR3-FR4 combination used was:

(SEQ ID NO: 96)      CDR3 NRGDIYYDFTY AFDIW GQGTMVTVSS(FR4 = JH3) 

For VH3 libraries, the CDR3-FR4 combination used differed by a singleamino acid in CDRH3:

(SEQ ID NO: 97)      CDR3 NRGDIYYDFTY A M DIWG QGTMVTVSS(FR4 = JH3) 

For light-chain repertoires, human Vkappa or Vlambda sequencescomprising FR1-CDRL1-FR2-CDRL2-FR3 were inserted upstream of selectedCDRL3-FR4 sequences. The CDRL3 comprises a binding specificitydeterminant from Mab 166 light-chain with the sequence FWXTP (where Xmay be S or G; SEQ ID NO:39). For Vkappa libraries, the C-terminalresidues of CDRL3 and FR4 were contributed by the human germ-line JK2sequence YTFGQGTKLEIK (SEQ ID NO:115; JK2 residues within CDRL3 areunderlined). For Vlambda libraries, the FR4 region was contributed byJL2 germ-line sequence FGGGTKLTVL (SEQ ID NO:48). The JL2 germlinesequence is identical to the JL3 sequence.

In some cases cassette libraries were constructed as described in USpatent application 20060134098 (library 1070). For library 1080,full-length lambda chains were screened in combination with VH cassettelibraries.

Heavy and light chain polypeptides were expressed as mature proteins,i.e., without a signal peptide, and secreted in E. coli cells thatexpress a mutant SecY gene as described in US patent application20070020685. The peptides therefore were expressed with an N-terminalmethionine. Binding of recombinant Fabs to PcrV was identified by afilter-binding assay using nitrocellulose filters coated with GST-PcrVfusion protein as described in US patent application 20050255552.Binding activity was confirmed by antigen ELISA using plates coated withGST-PcrV and affinities were determined by biolayer interferometry usinga ForteBio Octet biosensor.

The sequences of the V-regions of exemplary high-affinity anti-PcrVantibodies are shown in FIG. 1 and FIG. 2.

Each of the Fabs has high affinity for PcrV. Several Fabs wereidentified with affinities at least equivalent to Mab166 Fab(approximately 1.4 nM) determined by biolayer interferometry using aForteBio (Mountain View, Calif.) Octet biosensor.

VH and VL regions identified as described can be used in variouscombinations. For example, a V_(K) light chain SEQ ID NO:12 supportshigh affinity binding to PcrV in combination with either a V_(H)comprising SEQ ID NO:11, or a V_(H) comprising SEQ ID NO:3.

The 1070-9E antibody is an example of a high affinity antibody derivedby V-region cassette exchange using methods described in US PatentApplication Publication NO. 20060134098. To isolate this antibody, 4V-region replacement cassettes were constructed: 1) heavy chainfront-end cassette (consisting of human VH3 FR1-CDR1-FR2 sequences) 2)heavy chain middle cassette (consisting of human VH3 FR2-CDR2-FR3sequences) 1) light chain front-end cassette (consisting of human VK1FR1-CDR1-FR2 sequences) 2) light chain middle cassette (consisting ofhuman VK1 FR2-CDR2-FR3 sequences). Each cassette was assembled withadditional V-region sequences from Mab 166 and the selected CDR3-FR4region and expressed as Fab fragments in E. coli TOP10 cells transformedwith a plasmid over-expresssing a mutant SecY gene to allow secretion ofsignal-less Fabs. Cassette Fab libraries were then screened on GST-PcrVcoated filters to identify PcrV binders. Selected sequences from Fabssupporting PcrV binding were then recombined and re-screened to identifyfully-human V-segments supporting high-affinity binding to PcrV.

Fab 1070-9E, isolated by cassette recombination, has an affinity forrecombinant PcrV of 1.48 nM, determined by biolayer interferometry.

High-affinity anti-PcrV Fabs are also potent antagonists of the P.aeruginosa Type III Secretion system and inhibit P. aeruginosaexotoxin-mediated killing of P3-X63 Ag8 myeloma cells by P. aeruginosastrain PA103 in a cell-based cytotoxicity assay.

Example 2 PEGylated Fab′

In this example, a Fab′ consisting of a human Fd′ heavy chain of theIgG1 sub-class and human kappa light chain linked by an inter-chaindisulfide bond involving the C-terminal cysteine of the kappa chain andthe cysteine residue C227 of the heavy chain (numbering sequentiallyfrom the N-terminus of the mature protein) was PEGylated. Therecombinant Fd′ heavy chain contains the IgG1 CH1 domain and the IgG1hinge region including two cysteine residues which are available afterreduction for conjugation to maleimide groups. Thus the expressedantibody protein is a disulfide-linked heterodimer of Fd′ heavy chainand a kappa light chain, containing a total of 452 amino acids. Thesequence of the constant region of an exemplary Fd′ is shown in FIG. 3.

To generate an immunoconjugate with a reduced rate of in vivo clearanceand thus an improved pharmacokinetic profile, the Fab′ is conjugated topolyethylene glycol (PEG). In di-PEGylated Fab′, each molecule of Fab′is conjugated to two long-chain PEG molecules by site-specificattachment at the hinge region exploiting the two available reactivethiols on the hinge cysteine residues and a maleimide derivatized PEG,methoxy-polyethylene glycol maleimide (mPEG-mal). The mPEG-mal moleculesare conjugated via thioether linkages between the maleimide moiety andthe hinge cysteine residues.

To generate di-PEGylated Fab′, mPEG-mal with average molecular weight of30 kD was obtained from NOF Corporation. The Fab′, which was expressedand secreted from E. coli, was prepared at a concentration of 4 mg/ml insodium citrate buffer pH 6.5 with 2 mM EDTA. Reducing agent (10 mM MEAat pH 6.5) was added for 30 minutes at room temperature and the reactionmixture was immediately desalted using a Zeba Desalt column (Pierce)pre-equilibrated with 10 mM glycine (pH 3) and 2 mM EDTA. mPEG-mal wasadded for 1 hour at room temperature and di-PEGylated Fab′ was separatedfrom other PEGylated species and from unreacted Fab′ using a HiTrap SPsepharose column on an Akta purification system from GE Healthcare.

The structure of di-PEGylated Fab′ is provided schematically in FIG. 4.The exemplary di-PEGylated Fab′ PEGylated in this example binds withhigh affinity to PcrV (affinity of 0.6 nM determined by surface plasmonresonance analysis) and is a potent antagonist of the P. aeruginosa TypeIII Secretion System.

Mono-PEGylated Fab′ can be generated using mutant derivatives of Fab′containing only a single hinge cysteine. The sequences of the mutanthinges are:

Wild-type human THTCPPCPA (SEQ ID NO: 112) gamma-1 hinge Mutant Hinge-1THT A PPCPA (SEQ ID NO: 113) Mutant Hinge-2 THTCPP A PA (SEQ ID NO: 114)

Example 3 Cytotoxicity Assay for Detection of Antibodies and FabFragments With Potent Neutralization Activity Against the P. AeruginosaType III Secretion System

A TTSS-dependent cytotoxicity assay was established using P3-X63-Ag8(X63) mouse myeloma cells (ATCC) as the target. Cells were cultured inRPMI 1640 (Media Tech) with 10% FBS (Hyclone). About 10⁵ cells wereinfected with P. aeruginosa strain PA103 at a multiplicity of infection(MOI) of 10 in a volume of 0.1 ml culture medium in wells of a 96-wellplate in the presence of Fab. Prior to addition of Fab and mammaliancells, PA103 was grown in MinS medium (Hauser A R et al. (1998) InfectImmun. 66:1413-1420) to induce expression of the TTSS. After incubationfor three hours at 37° C. with 5% CO2, with various concentrations ofanti-PcrV Fab, cells were transferred to 12×75 mm flow-cytometry tubesand stained with propidium iodide (Sigma) according to themanufacturer's instructions. The proportion of permeabilized cells wasquantified by flow cytometry using a FACS Caliber flow cytometer. Datawere analyzed using Prism4 software (Graphpad). (Cytotoxicity wasnormalized to dead cells in untreated samples). For comparison of thepotency of different Fabs, mean concentrations required for 50%inhibition (IC₅₀) were obtained from at least 3 independent assays.Results for several exemplary Fabs are shown in Table 2 below.

TABLE 2 Potency of Fabs in cytotoxicity assay Fab IC₅₀ (nM) Mab166 Fab53.0 SEQ ID NOS: 13, 4 20.0 SEQ ID NOS: 13, 37 12.0 SEQ ID NOS: 5, 650.2 SEQ ID NOS: 13, 10 25.5 SEQ ID NOS: 3, 4 35.1 SEQ ID NOS: 24, 2625.5 SEQ ID NOS: 35, 36 61.4

Each of the Fabs tested shows potent neutralization of the TTSS andprotection of mammalian cells from cytotoxicity.

Several Fabs are more potent in this assay than Mab 166 Fab. Thus,anti-PcrV antibodies of the invention typically show enhanced potencyrelative to Mab 166 Fab.

Example 4 Effects of an Antibody of the Invention in Vivo Using a MouseModel of Pneumonia

Experiments were performed in vivo using humaneered Fabs to evaluate theeffects of the antibodies in a mouse model of pneumonia. Fab 1A8 has ahuman VH3 sub-class heavy chain, containing the first constant domain ofhuman IgG1, and a human VKI sub-class kappa light chain. The affinity ofFab 1A8 as determined by Biacore is 0.6 nM. Fab 1A8 binds to PcrV withapproximately two-fold higher affinity than Mab 166 Fab.

An acute lethality model of Pseudomonas pneumonia was used to assess thein vivo efficacy of Fab 1A8 in comparison with Mab 166. P. aeruginosastrain PA103 was instilled directly into the lungs of mice at a dose of1.5×10⁶ cfu/mouse by intratracheal administration, an inoculum shownpreviously to be sufficient to lead to lethality in 100% of the animals(3×LD₉₀) (Sawa et al., Nat Med. 5:392-8, 1999). Survival and bodytemperature were monitored for 48 hours and surviving mice at this timepoint were sacrificed for determination of bacterial counts in thelungs. The survival data (FIG. 5) indicated that both the human Fab 1A8and the murine Fab can prevent lethality caused by the highly cytotoxicPA103 strain. Control mice infected with PA103 and treated with anirrelevant control Fab, were all dead within 24 hours of inoculation.Treatment of mice with 10 μg Mab 166 or Fab 1A8 led to the survival of100% of the mice at 48 hours. Since Fab 1A8 lacks the antibodyFc-region, antibody effector functions are not required for preventionof lethality. Fab 1A8 was significantly more potent than Mab166 Fab inprevention of lethality. Fab 1A8 provided significant protection fromlethality at doses of 1.25 μg and 0.625 μg/mouse, doses at which mouseMab166 Fab-treated animals showed 100% mortality (P<0.05 for differencesbetween Fab 1A8 and Mab 166 Fab at 2.5 μg, 1.25 μg and 0.625 μg doses).The activity of Fab 1A8 is comparable to that of Mab166 IgG inprevention of lethality.

Fab 1A8 is also effective in inducing recovery of body temperature,indicative of protection from sepsis (FIG. 6). Untreated mice infectedwith PA103 show a rapid drop in body temperature within the first fewhours of infection. Recovery of body temperature within 12-24 hours inthe antibody-treated groups correlates with subsequent survival. Dosesas low as 1.25 μg/mouse of Fab 1A8 or Mab 166 led to rapid recovery ofbody temperature and prevented lethality in at least 80% of mice.However, this dose of mouse Mab 166 Fab fragment was insufficient toallow body temperature recovery and all mice in this group were dead at48 hours post-infection.

Surviving mice at 48 hours post-challenge were also analyzed for thepresence of residual Pseudomonas in the lungs. Remarkably, both Mab 166and the Fab 1A8 fragments analyzed stimulated significant clearance ofbacteria (FIG. 7). After 48 hours, the bacterial counts were reduced atleast 1000-fold from the infectious dose of 1.5×10⁶ cfu/mouse in allmice treated with 10 μg Fab 1A8. 80% of mice treated with this dose ofFab 1A8 showed no detectable Pseudomonas in the lungs after 48 hours.Higher residual bacterial counts were detected in mice treated withmouse Mab 166 Fab. Human Fab 1A8 has comparable potency to the whole IgGMab 166 in this analysis indicating that Fc-effector functions do notcontribute significantly to the ability of the antibody to stimulatebacterial clearance.

A second humaneered Fab that has the Mab 166 minimal essential bindingspecificity determinant was also evaluated in vivo using a mouse modelof pneumonia. Female Balb/c mice (approximately 20 g in weight; CharlesRiver) were inoculated with 1×10⁶ P. aeruginosa strain PA103 byintra-tracheal administration. Prior to inoculation, PA103 bacteria weregrown overnight in YPT broth at 37° C., diluted 1:5 in fresh medium andgrown for two hours at 37° C. until they reached exponential phase. Theculture was centrifuged at room temperature for ten minutes at 2000 xgand the pellet resuspended in ˜8 mL phosphate buffered saline (PBS).Bacteria were quantified by absorbance at 600 nm and bacterialcolony-forming units verified by colony growth on tryptic soy (TS) agarplates (Teknova, Half Moon Bay, Calif.). Antibody Fab 2 fragment waspremixed with bacteria immediately prior to intratracheal instillation.Infected mice were monitored for body temperature (rectal temperatures)and survival for 48 hours.

Control mice treated only with saline solution showed 100% mortalitywithin 24 hours of bacterial inoculation. Mice treated with 10 μg Fab 2showed complete protection from lethality; 100% of the Fab-treated micesurvived at 48 hours.

This example thus shows that humaneered antibodies of the inventionexhibit potent in vivo activity against P. aeruginosa. The Fabs are morepotent than a parent M166 Fab in vivo.

Example 5 Evaluation of a Humaneered Fab for Immunogenicity in Human

A humaneered antibody PEGylated Fab′ fragment was evaluated for safety,immunogenicity and plasma/serum half-life in human subjects. Subjectsreceived one dose by intravenous (i.v.) injection at 1, 3, or 10 mg/kg.

The humaneered antibody was well tolerated at all dose levels. Theconcentration of drug in the plasma was measured by ELISA using the PcrVantigen immobilized onto a microtiter plate. GST-PcrV was immobilizedonto a microtiter plate overnight at 4° C. The plate was washed and allunadsorbed sites blocked with the addition of block/diluent buffer forat least 60 minutes. After washing the plate, analytes were dispensedonto the pre-coated microtiter plate and incubated for at least 60minutes. The plate was washed and a solution containing a biotinylatedantibody specific to the humaneered Fab was added for 45 minutes. Theplate was washed and a HRP-conjugate solution added for 30 minutes.After the final wash step, a tetramethylbenzidine (TMB) peroxidasesubstrate solution was added and incubated for approximately 6 minutes.The reaction was stopped with a phosphoric acid solution. Color developsin proportion to the amount of pegylated Fab present. Plates were readon a plate reader using two filters (450 nm for detection and 620 nm forbackground). Concentrations were determined on a standard curve obtainedby plotting optical density (OD) versus concentration. The calibrationcurve was generated using a four-parameter logistic fit. The range forthis method in human serum is from 0.200 to 12.8 ng/mL in 1% serum (20.0ng/mL to 1280 ng/mL in 100% serum).

Pharmacokinetic Profile of Humaneered Antibody in Human Subjects PK 1.0mg/kg 3.0 mg/kg 10.0 mg/kg Parameter Units n Cohort 1 Cohort 2 Cohort 3AUC(0-t) ng * hr/mL 4 10440826 (1137824) 33973664 (2930669) 120424224(17896301) AUC(0-∞) ng * hr/mL 4 10737696 (1235316) 34856666 (3216244)124429066 (19035293) % Extrap (%) 4  2.72 (0.886)  2.49 (0.935)  3.17(0.461) Cmax (ng/mL) 4 29334 (2039)  93533 (10738) 347287 (64571) T½(hr) 4   341 (38.5)   310 (37.9)   338 (14.1) CL (L/hr) 4  0.00693(0.000823)  0.00556 (0.000573)  0.00473 (0.00112) Vz (L) 4  3.44 (0.730) 2.50 (0.504)  2.31 (0.588)

The humaneered antibody had a terminal plasma half-life of approximately14 days.

The presence of anti-drug antibodies, i.e., antibodies generated to thehumaneered antibody, was tested at: pre-infusion, day 8, day 15 day 29and day 70 post infusion. Anti-drug antibodies were measured using anelectrochemiluminescent assay (ECLA). Positive controls and negativecontrol serum were diluted 1:25 with diluent buffer. The controls werefurther diluted 1:2 by the addition of an equal volume of 0.8% aceticacid (resulting in 2× solutions) and then incubated at ambienttemperature for approximately 15 minutes. Samples were then diluted anadditional 1:2 with Label Master Mix (Antibody-Biotin andAntibody-SulfoTag at 0.5 μg/mL final working concentrations) resultingin a final 1:100 dilution. All controls were then incubated for one hourat room temperature with gentle shaking. The Streptavidin-coatedstandard MA2400 96-well microtiter plate was blocked by adding diluentbuffer for 60 minutes. Diluent buffer was removed from plate wells byaspiration and controls were added to the plate and incubated for 60minutes. The plate was aspirated and washed, and 1× MesoScaleDiscovery®(MSD) Read Buffer T with surfactant was added. The plates were read onan MSD electrochemiluminescence detector within 1 minute. Intensity ofrelative light units (RLU) produced are in proportion to the amount ofanti-drug antibody present.

No anti-drug antibodies were detected at any time point. This examplethus demonstrates that there was no detectable immunogenicity of thehumaneered antibody in humans.

The following provides an exemplary listing of anti-PcrV antibodyV-regions of the invention:

Exemplary Anti-PcrV V-regions Vh (VH1) SEQ ID NO: 1EIQLVQSGAEVKKPGASVKVSCKASGYTFTDHAISWVRQAPGQGLEWMGWISPYSGNPNYAQSLQGRVSLTTDRSTRTAYMELRSLKSDDTAVYYCARNRGDIYYDFTYAFDIWGQGTM VTVSS VkISEQ ID NO: 2 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGRAPKLLIYAASSLQSGVPSRFSGSGSGTGFTLTISSLQPEDVATYYCQQFWSTPYTFGQGTKLEIK Vh SEQ ID NO: 3QVQLVESGGGVVQPGGSLRLSCAASGFTFSTAGMHWVRQAPGKGLEWVAVIWYNGKEISYADSVKGRFTVSRDNPKNTLYLQMSSLRTEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VkISEQ ID NO: 4 DIQMTQSPSSLSASVGDRVTITCRASQSISRWVAWYQQRPGKAPNLLIYDASSLKSGVPSRFSGSGSGTEFTLTISSLQPEDIATYYCQQFWSTPYTFGQGTKLEIK Vh SEQ ID NO: 5QVQLVESGGGVVQPGRSLRLSCTASGFSFSSYGMHWVRQAPGKGLEWVAVIWYNGKEISYADSVKGRFTVSRDNPKNTLYLQMSSLRTEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VkISEQ ID NO: 6 AIQLTQSPSFLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQFWSTPYTFGQGTKLEIK Vh SEQ ID NO: 7QVQLVESGGGLVQPGRSLRLSCVGSGFTFSSYGIHWVRQAPGKGLEWVAVIWYNGKEISYADSVKGRFTVSRDNLKNTLYLQMSSLRTEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VkISEQ ID NO: 8 DIQMTQSPSFLSASVGDRVTITCRASQGISTYLAWYQQKRGKAPKLLISAASSLQSGVPSRFSGSVSGTDFTLTISSLQSEDFAVYYCQQFWSTPYTFGQGTKLEIK Vh SEQ ID NO: 9QVQLVESGGGLVQPGRSLRLSCVGSGFTFSSYGIHWVRQAPGKGLEWVAVIWYNGKEISYADSVKGRFTVSRDNPKNTLYLQMSSLRTEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VkISEQ ID NO: 10DIQLTQSPSFLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKLLIYDASALQSGVPSRFSGSGSGTEFTLTISSLQPEDVATYYCQQFWSTPYTFGQGTKLEIK Vh SEQ ID NO: 11EVQLVESGGGVVQPGGSLRLSCAASGFTFSTAGMHWVRQAPGKGLEWVAVIWYNGKEISYADSVKGRFTVFRDNPKNTLYLQMSSLRTEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VkISEQ ID NO: 12DIQMTQSPSSLSASVGDRVTITCRASQSISRWVAWYQQRPGKAPNLLIYDASSLKSGVPSRFSGSGSGTEFTLTISSLQPEDIATYYCQQFWSTPYTFGQGTKLEIK Vh SEQ ID NO: 13QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYPLHWVRQAPGKGLEWVSFISYDGSEKYYASSVKGRFTISRDNSENTLYLQMNSLRPEDTAVYYCARNRGDIYYDFTYAMDIWGQGTMV TVSS VkSEQ ID NO: 14DIQLTQSPSFLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKLLIYDASALQSGVPSRFSGSGSGTEFTLTISSLQPEDVATYYCQQFWSTPYTFGQGTKLEIK Vh SEQ ID NO: 15EVQLVESGGGVVQPGRSLRLSCTASGFSFSSYGMHWVRQAPGKGLEWVAVIWYDGRNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VkIIISEQ ID NO: 16EIVLTQFPGTLSLSPGERATLSCRASQNVGSAYLAWYQQKPGQAPRLLIYGASRRAPGIPDRFSGSGSGTDFTLTINRLEPEDFAVYYCQQFWSTPYTFGQGTKLEIK Vh SEQ ID NO: 17EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGYNKDYADSVKGRFTISRDNSKNTLYLQINSLRAEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VkIIISEQ ID NO: 18EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQFWSTPYTFGQGTKLEIK Vh SEQ ID NO: 19EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYPLHWVRQAPGKGLEWVSFISYDGSEKYYASSVKGRFTISRDNSENTLYLQMNSLRPEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VkIIISEQ ID NO: 20EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLFYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQFWSTPYTFGQGTKLEIK Vh SEQ ID NO: 21EVQLVESGGGLVQPGRSLRLSCVGSGFTFSSYGIHWVRQAPGKGLEWVANIWYDGSSESYIDSVKGRFTVSRDDSRNTVYLQMNSLRPEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VkIIISEQ ID NO: 22EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQFWSTPYTFGQGTKLEIK VH SEQ ID NO: 23EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYPMHWVRQAPGKGLEWVAVISYDGSEKWYADSVKGRFTISRDNSKNTLYLEMNSLRPEDTAVYYCARNRGDIYYDFTYAMDQWGQGT TVTVSS VkSEQ ID NO: 24DIQLTQSPSTLSASVGDSVTITCRASEGVDRWLAWYQQKPGRAPKLLIYDASTLQSGVPSRFSGSGSGTEFSLTISSLQPDDVATYYCQHFWGTPYTFGQGTKLEIK VH SEQ ID NO: 25EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYPMHWVRQAPGKGLEWVAVISYDGSEKWYADSVKGRFTISRDNSKNTLYLEMNSLRPEDTAVYYCARNRGDIYYDFTYAMDSWGQGTTVTVSS VHSEQ ID NO: 26EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYPMHWVRQAPGKGLEWVAVISYDGSEKWYSRADSVKGRFTIDNSKNTLYLEMNSLRPEDTAVYYCARNRGDIYYDFTYAMDIWGQGTTVT VSS VHSEQ ID NO: 35EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYPMHWVRQAPGKGLEWVAVISYDGSEKWYADSVKGRFTISRDNSKNTLYLEMNSLRPEDTAVYYCARNRGDIYYDFTYAMDYWGQGTTVTVSS VkSEQ ID NO: 36DIQLTQSPSTLSASVGDSVTITCRASEGVDRWLAWYQQKPGRAPKLLIYDASTLQSGVPSRFSGSGSGTEFSLTISSLQPDDVATYYCQHFWSTPYTFGQGTKLEIKV-regions of Exemplary Antibodies with Lambda light chain VhSEQ ID NO: 27EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYPLHWVRQAPGKGLEWVSFISYDGSEKYYASSVKGRFTISRDNSENTLYLQMNSLRPEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VlSEQ ID NO: 28QSALTQPASVSGSPGQSITISCTGTSSDYVSWYQQHPGKAPKLIIYDVTNRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCQHFWSTPYTFGGGTKLTVL Vh SEQ ID NO: 29EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYPLHWVRQAPGKGLEWVSFISYDGSEKYYASSVKGRFTISRDNSENTLYLQMNSLRPEDTAVYYCARNRGDIYYDFTYAMDIWGQGTM VTVSS VlSEQ ID NO: 30SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCQHFWSTPYTFGGGTKLTVLAdditional V_(L) regions: Vl SEQ ID NO: 32SSELTQDPAVSVALGQTVTITCQGDSLRSLYASWYQQKPGQAPVLVLYSKNSRPSGIPDRFSGSSSGNTASLTITGARAEDEADYYCQHFWSTPYTFGGGTKLTVL Vl SEQ ID NO: 34QSVLTQPPSASGSPGQSVTISCTGTSSDVGAYNYVSWYQQYPGKVPKLIIYEVTKRPSGVPDRFSGSKSGNTASLTVSGLRAEDEADYYCQHFWSTPYTFGGGTKLTVL VkI SEQ ID NO: 37DIQMTQSPSSLSASVGDRVTITCRASQSISRWVAWYQQRPGKAPNLLIYDASSLKSGVPSRFSGSGSGTEFTLTISSLQPEDIATYYCQQFWGTPYTFGQGTKLEIK

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All publications, accession numbers, patents, and patent applicationscited in this specification are herein incorporated by reference as ifeach was specifically and individually indicated to be incorporated byreference.

What is claimed is:
 1. A method of treating a P. aeruginosa infection ina patient, the method comprising administering a therapeuticallyeffective amount of an anti-PcrV antibody that selectively binds PcrV toa patient infected with P. aeruginosa, wherein the antibody has a K_(D)of less than 50 nM when assayed as a Fab and comprises: a V_(L) regionthat has a CDR3 having a sequence Q(Q/H)FWGTPYT (SEQ ID NO:41); a CDR1having a sequence RASEGVDRWLA (SEQ ID NO:69) or RASQSISRWVA (SEQ IDNO:68); and a CDR2 having a sequence, DASTLQS (SEQ ID NO:73) or DASSLKS(SEQ ID NO:71); and a V_(H) region that has a CDR3 having a sequenceNRGDIYYDFTYA(M/F)DX₁ (SEQ ID NO:42), wherein X₁ is I, Q, Y or S; a CDR1having a sequence NYPMH (SEQ ID NO:54) or SYPLH (SEQ ID NO:53); and aCDR2 having a sequence VISYDGSEKWYADSVKG (SEQ ID NO:57) orFISYDGSEKYYASSVKG (SEQ ID NO:56).
 2. The method of claim 1, wherein theV_(H) region comprises a FR4 that has at least 90% identity to the FR4region of the human JH3 or human JH6 segment and the V_(L) regioncomprises a FR4 that has at least 90% identity to the FR4 region of thehuman JK2 germline gene segment or at least 90% identity to the JL2germline sequence.
 3. The method of claim 2, wherein the FR4 of theV_(H) region has the sequence of the corresponding FR4 region of humangermline JH3; and the FR4 of the V_(L) region has the sequence of thecorresponding FR4 region of human germline Jκ2.
 4. The method of claim1, wherein the antibody is a Fab′.
 5. The method of claim 4, wherein theantibody is PEGylated.
 6. The method of claim 5, wherein the antibody isdi-PEGylated.
 7. The method of claim 1, further comprising administeringan antibiotic.
 8. The method of claim 1, wherein the anti-PcrV antibodyis administered by insufflation, intravenous injection, or sub-cutaneousbolus injection.
 9. The method of claim 1, wherein the anti-PcrVantibody retains at least 75% of the Type III secretion pathwayantagonist activity of Mab166.
 10. The method of claim 1, wherein thepatient has cystic fibrosis.
 11. The method of claim 1, wherein theV_(H) region has a CDR3 having a sequence NRGDIYYDFTYAMDI (SEQ IDNO:99), a CDR1 having a sequence NYPMH (SEQ ID NO:54) or SYPLH (SEQ IDNO:53); and a CDR2 having a sequence VISYDGSEKWYADSVKG (SEQ ID NO:57) orFISYDGSEKYYASSVKG (SEQ ID NO:56).
 12. The method of claim 11, whereinthe V_(H) region has a CDR3 having a sequence NRGDIYYDFTYAMDI (SEQ IDNO:99), a CDR1 having a sequence NYPMH (SEQ ID NO:54), and a CDR2 havinga sequence VISYDGSEKWYADSVKG (SEQ ID NO:57); or a CDR3 having a sequenceNRGDIYYDFTYAMDI (SEQ ID NO:99), a CDR1 having a sequence SYPLH (SEQ IDNO:53) and a CDR2 having a sequence FISYDGSEKYYASSVKG (SEQ ID NO:56).13. The method of claim 1, wherein the V_(L) region has a CDR3 having asequence QHFWGTPYT (SEQ ID NO:110), a CDR1 having a sequence RASEGVDRWLA(SEQ ID NO:69) or RASQSISRWVA (SEQ ID NO:68), and a CDR2 having asequence DASTLQS (SEQ ID NO:73) or DASSLKS (SEQ ID NO:71).
 14. Themethod of claim 13, wherein the V_(L) region has a CDR3 having asequence QHFWGTPYT (SEQ ID NO:110), a CDR1 having a sequence RASEGVDRWLA(SEQ ID NO:69) and a CDR2 having a sequence DASTLQS (SEQ ID NO:73); orthe V_(L) region has a CDR3 having a sequence QHFWGTPYT (SEQ ID NO:110),a CDR1 having a sequence RASQSISRWVA (SEQ ID NO:68), and a CDR2 having asequence DASSLKS (SEQ ID NO:71).
 15. The method of claim 1, wherein theV_(H) region has a CDR3 having a sequence NRGDIYYDFTYAMDI (SEQ IDNO:99), a CDR1 having a sequence NYPMH (SEQ ID NO:54), and a CDR2 havinga sequence VISYDGSEKWYADSVKG (SEQ ID NO:57); and the V_(L) region has aCDR3 having a sequence QHFWGTPYT (SEQ ID NO:1 10), a CDR1 having asequence RASEGVDRWLA (SEQ ID NO:69), and a CDR2 having a sequenceDASTLQS (SEQ ID NO:73).
 16. The method of claim 1, wherein the V_(H)region has a V-segment sequence of a V-segment of SEQ ID NO:13, 19, 23,25, 26, or 35; and the V_(L) region has a V-segment sequence of aV-segment of SEQ ID NO:24, 12, 4, or
 37. 17. The method of claim 16,wherein the V_(H) region has a sequence of SEQ ID NO:13, 19, 23, 25, 26,or 35; and the V_(L) region has a sequence of SEQ ID NO:24 or
 37. 18.The method of claim 17, wherein the V_(H) region has a sequence of SEQID NO:26 and the V_(L) region has a sequence of SEQ ID NO:24.